Functional Th1-oriented T follicular helper cells that infiltrate human breast cancer promote effective adaptive immunity
Grégory Noël, … , Denis Larsimont, Karen Willard-Gallo
Grégory Noël, … , Denis Larsimont, Karen Willard-Gallo
Published August 19, 2021
Citation Information: J Clin Invest. 2021;131(19):e139905. https://doi.org/10.1172/JCI139905.
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Research Article Immunology Oncology

Functional Th1-oriented T follicular helper cells that infiltrate human breast cancer promote effective adaptive immunity

Abstract

We previously demonstrated that tumor-infiltrating lymphocytes (TIL) in human breast cancer sometimes form organized tertiary lymphoid structures (TLS) characterized by CXCL13-producing T follicular helper (Tfh) cells. The present study found that CD4+ Tfh TIL, CD8+ TIL, and TIL-B, colocalizing in TLS, all express the CXCL13 receptor CXCR5. An ex vivo functional assay determined that only activated, functional Th1-oriented Tfh TIL (PD-1hiICOSint phenotype) provide help for immunoglobulin and IFN-γ production. A functional Tfh TIL presence signals an active TLS, characterized by humoral (immunoglobulins, Ki-67+ TIL-B in active germinal centers) and cytotoxic (GZMB+CD8+ and GZMB+CD68+ TIL plus Th1 gene expression) immune responses. Analysis of active versus inactive TLS in untreated patients revealed that the former are associated with positive clinical outcomes. TLS also contain functional T follicular regulatory (Tfr) TIL, which are characterized by a CD25+CXCR5+GARP+FOXP3+ phenotype and a demethylated FOXP3 gene. Functional Tfr inhibited functional Tfh activities via a glycoprotein A repetitions predominant (GARP)-associated TGF-β–dependent mechanism. The activity of tumor-associated TLS was dictated by the relative balance between functional Tfh TIL and functional Tfr TIL. These data provide mechanistic insight into TLS processes orchestrated by functional Th1-oriented Tfh TIL, including TIL-B and CD8+ TIL activation and immunological memory generation. Tfh TIL, regulated by functional Tfr TIL, are an expected key target of PD-1/PD-L1 blockade.

Authors

Grégory Noël, Mireille Langouo Fontsa, Soizic Garaud, Pushpamali De Silva, Alexandre de Wind, Gert G. Van den Eynden, Roberto Salgado, Anaïs Boisson, Hanne Locy, Noémie Thomas, Cinzia Solinas, Edoardo Migliori, Céline Naveaux, Hugues Duvillier, Sophie Lucas, Ligia Craciun, Kris Thielemans, Denis Larsimont, Karen Willard-Gallo

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Figure 1

CXCR5+ TIL are primarily localized in human BC-associated TLS.

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CXCR5+ TIL are primarily localized in human BC-associated TLS. (A) Lymph...
(A) Lymphocytes from fresh tissue homogenates (ref. 29) of human tonsils (n = 10) or BC (n = 168; luminal A [LumA; n = 82], luminal B [LumB; n = 36], HER2+ [n = 24], TN [n = 26]) were immunophenotyped (flow cytometry). The mean frequency (μ) of CXCR5+ cells within the CD4, CD8, and CD20 subpopulations is shown. Upper plots: CD3+CD4+CD45+ T cells; middle plots: CD3+CD8+CD45+ T cells; lower plots: CD19+CD45+ B cells. (B) TIL densities in human BC tumors (n = 168; number of TIL/mg of tumor; flow cytometry) for CXCR5+ Tfh TIL, CXCR5+ TIL-B, and CD8+CXCR5+ TIL correlated with one another (linear regression analysis). (C) TIL densities (CXCR5+Tfh TIL + CD8+CXCR5+ TIL + CXCR5+ TIL-B/mg of tumor; flow cytometry) were correlated with TIL aggregates or TLS scored on dual CD3/CD20 (brown/red) cIHC-stained FFPE tissue sections (as described in refs. 3, 39) from the same tumor (n = 79; linear regression analysis). (D) Upper panel: representative dual CD3/CD20 cIHC (TN BC 0989); lower panel: zoom images of 3 TLS areas: 1, inside a TLS showing the T cell zone and B cell follicle; 2 and 3, focused on areas outside the TLS. Upper panel: magnification ×3.5; lower panel magnification: ×35. (E) IF staining of a consecutive tissue section showing the 3 areas in D. Left panels: colocalization of CD20+CXCR5+ TIL-B (yellow) and CD4+CXCR5+ Tfh TIL (purple) or CD8+CXCR5+ TIL (purple) inside the TLS. Magnification × 60. Right panels: CXCR5– CD4+/CD8+ (blue) or CD20+ (green) TIL outside the TLS. The T:B border is the junction between the T cell zone and the B cell follicle. Upper right magnification ×100; lower right magnification ×150.